title: Establishment and application of high throughput screening model for hepatitis C virus NS3-4A protease inhibitors in vitro.
abstract: OBJECTIVE
  To establish fluorescence resonance energy transfer (FRET) assay method of detecting proteolytic activity of non-structural protein 3-4A (NS3-4A) serine protease of hepatitis C virus (HCV) for high throughput screening inhibitors against HCV in vitro.
  
  
  METHODS
  HCV recombinant plasmid pMAL~c2/NS3-4A was transformed into the E.coli strain K12TB1. Maltose-binding-protein (MBP) NS3-4A fusion protein expression was induced by adding isopropyl-β-D-thiogalacto-pyranoside (IPTG) and purified by affinity chromatography. The proteolytic activity of MBP-NS3-4A protease was analyzed by FRET with the special protease substrate. The reaction system in this model was optimized, and the reliability of the model was evaluated.
  
  
  RESULTS
  High throughput screening model for HCV NS3-4A protease inhibitors was established, and the best concentrations of enzyme and substrate were optimized. In the model, the Km value of protease was 4.74 μmol/L, Z factor was up to 0.80, and coefficient of variation (CV) was 1.91%. BILN 2061, one of the known HCV protease inhibitors, was measured with the Ki of 0.30 nmol/L.
  
  
  CONCLUSIONS
  The assay model using FRET method for HCV NS3 4A serine protease is stable and reliable, and the model is suitable for high throughput screening for HCV NS3 4A protease inhibitors.
authors.affiliation: Department of Virology, Institute of Medicinal Biotechnology, CAMS and PUMC, Beijing 100050, China.
authors.forename: Li
authors.initials: L
authors.lastname: Jian-rui
authors.forename: Wu
authors.initials: W
authors.lastname: Yan-bin
authors.forename: Si
authors.initials: S
authors.lastname: Shu-yi
authors.forename: Chen
authors.initials: C
authors.lastname: Hong-shan
authors.forename: Jiang
authors.initials: J
authors.lastname: Jian-dong
authors.forename: Bang
authors.initials: B
authors.lastname: Zong-gen
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D031541:Fluorescence Resonance Energy Transfer; D016174:Hepacivirus / Q000201:enzymology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D011480:Protease Inhibitors / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases